Betsy L. Lytle

Cell-free protein production and labeling protocol for NMR-based structural proteomics

Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods. To illustrate this platform, we describe its application to a specific target (At3g01050.1) from Arabidopsis thaliana. After cloning the target gene into a specialized plasmid, we carry out a small-scale (50 μl) in vitro sequential transcription and translation trial to ascertain the level of protein production and solubility. Next, we prepare mRNA for use in a 4-ml semicontinuous cell-free translation reaction to incorporate 15N-labeled amino acids into a protein sample that we purify and test for suitability for NMR structural analysis. We then repeat the cell-free approach with 13C,15N-labeled amino acids to prepare a doubly labeled sample. The three-dimensional (3D) structure of At3g01050.1 shows that this protein is an unusual member of the β-grasp protein family.

The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome.

Cellobiohydrolase CelS plays an important role in the cellulosome, an active cellulase system produced by the thermophilic anaerobe Clostridium thermocellum. The structures of the catalytic domain of CelS in complex with substrate (cellohexaose) and product (cellobiose) were determined at 2.5 and 2.4 A resolution, respectively. The protein folds into an (alpha/alpha)(6) barrel with a tunnel-shaped substrate-binding region. The conformation of the loops defining the tunnel is intrinsically stable in the absence of substrate, suggesting a model to account for the processive mode of action of family 48 cellobiohydrolases. Structural comparisons with other (alpha/alpha)(6) barrel glycosidases indicate that CelS and endoglucanase CelA, a sequence-unrelated family 8 glycosidase with a groove-shaped substrate-binding region, use the same catalytic machinery to hydrolyze the glycosidic linkage, despite a low sequence similarity and a different endo/exo mode of action. A remarkable feature of the mechanism is the absence, from CelS, of a carboxylic group acting as the base catalyst. The nearly identical arrangement of substrate and functionally important residues in the two active sites strongly suggests an evolutionary relationship between the cellobiohydrolase and endoglucanase families, which can therefore be classified into a new clan of glycoside hydrolases.

Orphan Macrodomain Protein (Human C6orf130) Is an O-Acyl-ADP-ribose Deacylase SOLUTION STRUCTURE AND CATALYTIC PROPERTIES

Background: Protein deacylation by sirtuins yields O-acyl-ADP-ribose molecules, which are implicated in cell signaling and metabolism. Results: Structural and functional analysis of a human orphan macrodomain protein reveals a common core structure and ability to hydrolyze O-acyl-ADP-ribose. Conclusion: Diverse macrodomains are capable of hydrolyzing O-acyl-ADP-ribose but utilize a different set of catalytic amino acids. Significance: Macrodomain-containing proteins provide a biochemical and biological link to sirtuin-dependent deacylation. Post-translational modification of proteins/histones by lysine acylation has profound effects on the physiological function of modified proteins. Deacylation by NAD+-dependent sirtuin reactions yields as a product O-acyl-ADP-ribose, which has been implicated as a signaling molecule in modulating cellular processes. Macrodomain-containing proteins are reported to bind NAD+-derived metabolites. Here, we describe the structure and function of an orphan macrodomain protein, human C6orf130. This unique 17-kDa protein is a stand-alone macrodomain protein that occupies a distinct branch in the phylogenic tree. We demonstrate that C6orf130 catalyzes the efficient deacylation of O-acetyl-ADP-ribose, O-propionyl-ADP-ribose, and O-butyryl-ADP-ribose to produce ADP-ribose (ADPr) and acetate, propionate, and butyrate, respectively. Using NMR spectroscopy, we solved the structure of C6orf130 in the presence and absence of ADPr. The structures showed a canonical fold with a deep ligand (ADPr)-binding cleft. Structural comparisons of apo-C6orf130 and the ADPr-C6orf130 complex revealed fluctuations of the β5-α4 loop that covers the bound ADPr, suggesting that the β5-α4 loop functions as a gate to sequester substrate and offer flexibility to accommodate alternative substrates. The ADPr-C6orf130 complex identified amino acid residues involved in substrate binding and suggested residues that function in catalysis. Site-specific mutagenesis and steady-state kinetic analyses revealed two critical catalytic residues, Ser-35 and Asp-125. We propose a catalytic mechanism for deacylation of O-acyl-ADP-ribose by C6orf130 and discuss the biological implications in the context of reversible protein acylation at lysine residues.

Structure of the B3 domain from Arabidopsis thaliana protein At1g16640

A novel DNA binding motif, the B3 domain, has been identified in a number of transcription factors specific to higher plant species, and was recently found to define a new protein fold. Here we report the second structure of a B3 domain, that of the Arabidopsis thaliana protein, At1g16640. As part of an effort to ‘rescue’ structural genomics targets deemed unsuitable for structure determination as full‐length proteins, we applied a combined bioinformatic and experimental strategy to identify an optimal construct containing a predicted conserved domain. By screening a series of N‐ and C‐terminally truncated At1g16640 fragments, we isolated a stable folded domain that met our criteria for structural analysis by NMR spectroscopy. The structure of the B3 domain of At1g16640 consists of a seven‐stranded β‐sheet arranged in an open barrel and two short α‐helices, one at each end of the barrel. While At1g16640 is quite distinct from previously characterized B3 domain proteins in terms of amino acid sequence similarity, it adopts the same novel fold that was recently revealed by the RAV1 B3 domain structure. However, putative DNA‐binding elements conserved in B3 domains from the RAV, ARF, and ABI3/VP1 subfamilies are largely absent in At1g16640, perhaps suggesting that B3 domains could function in contexts other than transcriptional regulation.

Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation.

Immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors inhibit cellular responsiveness to immunoreceptor tyrosine-based activation motif (ITAM)-linked receptors. Although tyrosine phosphorylation is central to the initiation of both inhibitory ITIM and stimulatory ITAM signaling, the events that regulate receptor phosphorylation are incompletely understood. Previous studies have shown that ITAM tyrosines engage in structure-inducing interactions with the plasma membrane that must be relieved for phosphorylation to occur. Whether ITIM phosphorylation is similarly regulated and the mechanisms responsible for release from plasma membrane interactions to enable phosphorylation, however, have not been defined. PECAM-1 is a dual ITIM-containing receptor that inhibits ITAM-dependent responses in hematopoietic cells. We found that the PECAM-1 cytoplasmic domain is unstructured in an aqueous environment but adopts an α-helical conformation within a localized region on interaction with lipid vesicles that mimic the plasma membrane. The lipid-interacting segment contains the C-terminal ITIM tyrosine and a serine residue that undergo activation-dependent phosphorylation. The N-terminal ITIM is excluded from the lipid-interacting segment, and its phosphorylation is secondary to phosphorylation of the membrane-interacting C-terminal ITIM. On the basis of these findings, we propose a novel model for regulation of inhibitory signaling by ITIM-containing receptors that relies on reversible plasma membrane interactions and sequential ITIM phosphorylation.

Solution structure of thioredoxin h1 from Arabidopsis thaliana

Present in virtually every species, thioredoxins catalyze disulfide/dithiol exchange with various substrate proteins. While the human genome contains a single thioredoxin gene, plant thioredoxins are a complex protein family. A total of 19 different thioredoxin genes in six subfamilies has emerged from analysis of the Arabidopsis thaliana genome. Some function specifically in mitochondrial and chloroplast redox signaling processes, but target substrates for a group of eight thioredoxin proteins comprising the h subfamily are largely uncharacterized. In the course of a structural genomics effort directed at the recently completed A. thaliana genome, we determined the structure of thioredoxin h1 (At3g51030.1) in the oxidized state. The structure, defined by 1637 NMR‐derived distance and torsion angle constraints, displays the conserved thioredoxin fold, consisting of a five‐stranded β‐sheet flanked by four helices. Redox‐dependent chemical shift perturbations mapped primarily to the conserved WCGPC active‐site sequence and other nearby residues, but distant regions of the C‐terminal helix were also affected by reduction of the active‐site disulfide. Comparisons of the oxidized A. thaliana thioredoxin h1 structure with an h‐type thioredoxin from poplar in the reduced state revealed structural differences in the C‐terminal helix but no major changes in the active site conformation.

The solution structure of ZNF593 from Homo sapiens reveals a zinc finger in a predominately unstructured protein

Here, we report the solution structure of ZNF593, a protein identified in a functional study as a negative modulator of the DNA‐binding activity of the Oct‐2 transcription factor. ZNF593 contains a classic C2H2 zinc finger domain flanked by about 40 disordered residues on each terminus. Although the protein contains a high degree of intrinsic disorder, the structure of the zinc finger domain was resolved by NMR spectroscopy without a need for N‐ or C‐terminal truncations. The tertiary structure of the zinc finger domain is composed of a β‐hairpin that positions the cysteine side chains for zinc coordination, followed by an atypical kinked α‐helix containing the two histidine side chain ligands. The structural topology of ZNF593 is similar to a fragment of the double‐stranded RNA‐binding protein Zfa and the C‐terminal zinc finger of MBP‐1, a human enhancer binding protein. The structure presented here will provide a guide for future functional studies of how ZNF593 negatively modulates the DNA‐binding activity of Oct‐2, a POU domain‐containing transcription factor. Our work illustrates the unique capacity of NMR spectroscopy for structural analysis of folded domains in a predominantly disordered protein.

Structures of proteins of biomedical interest from the Center for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) produces and solves the structures of proteins from eukaryotes. We have developed and operate a pipeline to both solve structures and to test new methodologies. Both NMR and X-ray crystallography methods are used for structure solution. CESG chooses targets based on sequence dissimilarity to known structures, medical relevance, and nominations from members of the scientific community. Many times proteins qualify in more than one of these categories. Here we review some of the structures that have connections to human health and disease.

Solution structure of Arabidopsis thaliana protein At5g39720.1, a member of the AIG2-like protein family

The three-dimensional structure of Arabidopsis thaliana protein At5g39720.1 was determined by NMR spectroscopy. It is the first representative structure of Pfam family PF06094, which contains protein sequences similar to that of AIG2, an A. thaliana protein of unknown function induced upon infection by the bacterial pathogen Pseudomonas syringae. The At5g39720.1 structure consists of a five-stranded beta-barrel surrounded by two alpha-helices and a small beta-sheet. A long flexible alpha-helix protrudes from the structure at the C-terminal end. A structural homology search revealed similarity to three members of Pfam family UPF0131. Conservation of residues in a hydrophilic cavity able to bind small ligands in UPF0131 proteins suggests that this may also serve as an active site in AIG2-like proteins.

Solution structure of a membrane-anchored ubiquitin-fold (MUB) protein from Homo sapiens

The protein Bc059385, whose solution structure is reported here, is the human representative of a recently identified family of membrane‐anchored ubiquitin‐fold (MUB) proteins. Analysis of their similarity to ubiquitin indicates that homologous amino acid residues in MUBs form a hydrophobic surface very similar to the recognition patch surrounding Ile‐44 in ubiquitin. This suggests that MUBs may interact with proteins containing an α‐helical motif similar to those of some ubiquitin binding domains. A disordered loop common to MUBs may also provide a second protein interaction site. From the available data, it is probable that this protein is prenylated and associated with the membrane. With <20% identity to ubiquitin, the MUB family further expands the sequence space that maps to the β‐grasp fold, and adds membrane localization to its list of functional roles.